Our recent work has focused upon studies of the internal motions of the HIV-1 protease (a) free in solution, as a fully active, but stable protease mutant (Q7K, L33I, L63I) (b) complexed with DMP323, a symmetric, specific and potent (Ki ~ 1 nM) inhibitor, and (c) complexed with KNI529, a potent (Ki ~ 1 nM) asymmetric inhibitor. We have developed a novel method to study slow, biologically significant, motions in proteins using complementary measurements of amide 15N and 1H transverse relaxation times. These measurements revealed that in the free protease, residues G48 through I54 (in the flaps of the protein) undergo slow conformation exchange on the ms time scale. In most crystal structures of the free protease, the flaps form beta-hairpin structures that are not fully closed over the active site. We suggest that the fast nanosecond motions of the flaps, that we have also identified, involve conformational fluctuations among these semi-open hairpin conformations, whereas the slow motions reflect the dynamic equilibrium between the semi-open and fully open flap conformations. It is these latter, slow conformational fluctuations of the protease flaps that permit entry of substrates and inhibitors to the active site of the protein. Because KNI-529 is an asymmetric inhibitor that is in slow exchange with the protein, the protease monomers have distinct NMR spectra when the protein is bound to the inhibitor. The rate of monomer exchange, k, was determined by measuring the diagonal and cross peak intensities of the protease amide NH signals as a function of the NOESY mixing time. It was found that k increased from ca. 0.2 s-1 to 6 s-1 upon increasing temperature from 25 to 45 C. Our data together with calorimetric measurements show that the inhibitor flips without dissociating from the protein. This result and our rate measurements imply an activation enthalpy of 32 kcal M-1 is for the reaction. The protease flaps must open in order for the inhibitor to flip, and we estimate that the rate of flap opening is ca.1 s-1 at 35 C based upon our measurements of k (the monomer exchange rate) discussed above. It is noteworthy that the rate of flap opening is ca. three-four orders of magnitude slower in the presence of KNI529 than in the free protease.